Decyder 7

For the 2D-DIGE experiment, DeCyder 7.0 (GE Healthcare) was used to analysis data from DIGE (DIA and BVA model). For Western blot data, One-way ANOVA with SPSS version 13.0 (SPSS Inc., Chicago, IL, United States) was used for the analysis of the data. The differences between the groups were analyzed by Bonferroni’s post hoc test. All data were shown as mean ± SD and the control group was normalized to 100%. Prism software (GraphPad Prism 5, La Jolla, CA, United States) was used to create the pictures. A value of p< 0.05 was considered to be statistically significant.

All open-source and commercial software and proteomic databases used to analyse MS/MS data, RNA-sequencing data and microscopy pictures are described in the Methods. All statistical analyses were performed using Stata v.15.1 and GraphPad Prism v.10. Image analysis was performed with Fiji/ImageJ 2.00/1.52p and ZEN Pro 3.1. The 2D gel captures were analysed with DeCyder 7.0 and ImageQuant (GE Healthcare). Pulsed SILAC data were analysed with Qlucore Omics Explorer 3.5 software. FACS data were analysed with FlowJo v.10. Gene set enrichment analyses were performed with GSEA v.4.3.2. The 3D molecular structure of the PPIA protein was visualized with PyMOL v.2.5.2 (licensed by A.C.). Figures 1c,g,2a,4a and 6d were created with BioRender.com (licensed by L.M.).

Gels were scanned with the Typhoon 9400 scanner (GE Healthcare) at the wavelengths corresponding to each CyDye. As explained in the previous sub-section, 27 gel images were obtained for the mitoproteome analysis (3 conditions: WT, LE, HE) and 18 for the other analyses (2 conditions: WT and HE). Gels images were analyzed with the DeCyder 7.0 software (GE Healthcare). Briefly, codetection of the three CyDye-labeled forms of each spot was performed using the DIA (Differential In-gel Analysis) module. The DIA module performs the spot detection, the ratio calculation, and the spot abundance normalization via the internal standard. Statistical analyses were carried out in the BVA (Biological Variation Analysis) module after intergel matching. Protein spots that showed a statistically significant Student’s t-test (p ≤ 0.05, n = 6) and ANOVA-1 (p ≤ 0.05, n = 6) for an increase or decrease ranging up to +1.2 or down to -1.2 in normalized ratio intensity were accepted as being differentially expressed between wild-type and HTG mice. In order to ensure relevant quantitation and avoiding any bias due to the extraction methods, Student’s t-test applied to matrix and inner-membrane 2D-DIGE comparisons were considered as significant when ≤ 0.01. Statistical analyses for the re-normalization procedure were performed by using Excel (Microsoft) and JMP 10 (SAS) software.

Before spot detection, acquired images were warped using Robust Automated Image Normalization (13 (link), 14 (link)) applying the recommended standard settings. All gels were aligned to the IPS (S-300 channel) of Gel01 (Figure S1). Protein spot detection was carried out using DeCyder 7.0 (GE Healthcare, RRID: SCR_014592). On average 2,359 (range: 2,174–2,715) spots per gel were detected. The normalized spot volumes were exported using the DeCyder XML toolbox (GE Healthcare), standardized against the spot volume of the corresponding spot in the IPS (S-300) channel and log10 transformed. Data was analyzed using R (version 3.6.2, The R Foundation for Statistical Computing, RRID: SCR_001905) (15 ) using the additional libraries plyr (16 (link)) and reshape (17 (link)).

We used the Zoom IPGRunner system (Invitrogen) to separate proteins in two dimensions. We isolated the HSPCs of young male and female C57BL/6J mice (four to eight months of age) and lysed them according to the manufacturer’s instructions with urea, CHAPS, dithiothreitol (DTT) and ampholytes. CyDyes DIGE fluors (minimal dyes) were used according to the vendor’s instructions (Amersham) with fluorophores Cy3 and Cy5 for post-lysis labelling to ensure that only 1–2% of lysines were labelled in a given protein. Labelling intensities were measured with a Typhoon FLA 9000 scanner and quantified with DeCyder 7.0 and ImageQuant software (GE Healthcare). We normalized the total protein abundance based on protein size and lysine concentration for spots with known identity by MS/MS. PPIA quantity was identical in two independent experiments using different fluorophores.

The standard of 2D-DIGE analysis was performed according to the protocol described previously (Dowling and Ohlendieck, 2018 (link)). Most importantly, to reduce the variation of signal in gels, the photomultiplier tube (PMT) was set to ensure maximum pixel intensity values for all gel images within a range of 40,000–60,000 pixels. DeCyder 7.0 (GE Healthcare) was used to analyze the images. In the DIA module, each spot was detected, matched, and normalized; in the BVA module, spot statistics were reviewed. The spots with an average ratio of more than +1.5 or less than −1.5 and with a statistical difference (p < 0.05) were isolated for further investigation.

Latex proteins were extracted as described33 (link). Protein concentration was determined by Bradford assay with BSA as a standard. For common 2-DE, 1,000 μg of proteins were loaded onto the 24-cm, pH 4-7 linear gradient IPG strips (GE Healthcare, Uppsala, Sweden), and then, isoelectric focusing and gel electrophoresised were performed as described19 64 (link). Three biological replicates for each separation were conducted.
2-D DIGE analysis was performed as described64 (link). Proteins from the D-3, E-3, D-5 and E-5 latex samples were labeled with a ratio of 400 pmol Cy3 or Cy5 per 50 μg of proteins. For gel normalization, an internal standard was prepared by pooling an equal protein quantity from each of the four samples and labeling with Cy2. Then, the Cy2-, Cy3- and Cy5-labeled 2-DE images were acquired using a Typhoon Trio scanner (GE Healthcare, Piscataway, NJ, USA), and the DIGE images were analyzed using DeCyder 7.0 software (GE Healthcare). A differential in-gel analysis module was used for spot detection, and a biological variation analysis module was applied to the three biological repeats to identify the differentially expressed spots with higher than 95% confidence.